The present invention relates generally to fluorescence imaging, and more particularly to field flattening in fluorescence imaging.
Fluorescence imaging typically includes illuminating a dye-labeled target with light having wavelength content that matches, at least partially, the absorption spectrum of a fluorescence dye and imaging the target with an optical system that favors the emitted fluorescence light over any reflected or scattered portion of the excitation light. As with any other fluorescence imaging system the amount of light that reaches the image plane, and hence a detector, for a given amount of fluorescent dye concentration at the target plane varies spatially. The three key components for this variation can be expressed by the following equation:SPixel(P,t,x,y)=[SFl(P,t,x,y)+SScatter(P,t,x,y)]
where Spixel (P,t,x,y) is the amount of light arriving at a pixel of a detector such as a CCD. SFl(P,t,x,y) is the fluorescence signal from a desired dye-labeled target and varies at low concentrations linearly with the power of the excitation light, P, detector exposure time, t, and varies spatially with the excitation spatial distribution as well as the response of the imaging system, x and y. Sscatter(P,t,x,y) is the amount of scattered light reaching a pixel comprised primarily of excitation light varying linearly with excitation light, P, detector exposure time, t, and can vary spatially with the excitation spatial distribution, system components scattering excitation light, etc. as well as the response of the imaging system, x and y. This scattered light could also encompass other sources of light such as photoluminescence of objects/materials in the instrument that exhibit auto-fluorescence and/or scatter light when exposed to the excitation light. Other contributions that affect the spatial variation of light impinging on a pixel is the typical radiative falloff that all imaging systems experience of cos4 θ, where θ is the angle between image point, or pixel, and the optical axis (center line through the optical system) and is easily corrected for with an electronics detector array such as a CCD-based imaging system. Vignetting is another factor that, if present, will also impact the pixel signal value whose influence would exhibit typically as a faster rate of radiative falloff in the image plane with increasing θ.
To accurately quantitate a desired dye-labeled target at some concentration the image pixel signal reported needs to be constant for that amount of fluorescent dye everywhere across the field of view. Thus, the same signal level is reported whether the target is placed at the center of the object plane or at the extremities of the imaged field of view. This requires knowledge of the imaging system's spatial signal response. Normally the fluorescence signal dominates and can be limited in detectability by the electrical background of the detection system at short exposure lengths. At longer exposure lengths, the electrical noise becomes smaller than the scattered light signal allowing the spatial variation of this component to be observed. At these longer exposure lengths, pixel signal is dominated by fluorescence at higher light levels with scattered light becoming dominant at lower light levels resulting in a non-uniform spatial system response.
Spatial variation of the fluorescence light can be minimized by employing a methodology for obtaining uniform excitation illumination of the target area over the object plane of the imaging system as described in patent application publication 2009/0080194 A1, which is hereby incorporated by reference. Minimizing the contribution of scattered light can be accomplished by ensuring the imaging or detection system efficiently filters out excitation light. This can be achieved by employing a filtering strategy as described in U.S. Pat. No. 7,286,232, which is hereby incorporated by reference. Employing good practices as these will tend to result in the fluorescence spatial variation to be radially symmetric about the center of the image. However, the spatial variation due to scatter will likely not be symmetric, depending more on system configuration and components that light scatters from.
Nonetheless, it is desirable to provide systems and methods for flattening the image across the entire field by correcting the image for both the fluorescence and scatter spatial variations.
Therefore it is desirable to provide systems and methods that overcome the above and other problems.